A certain species of enzymes in living body have serine, threonine, or tyrosine residue in a specific site such as an active center or an allosteric site. The activity of these enzymes is controlled by phosphorylation or dephosphorylation of those hydroxyl groups by enzyme called kinase. In addition, in some enzymes, the activity thereof is controlled by phosphorylation or dephosphorylation of an amino group or an imino group of lysine, arginine, or histidine, or a carboxyl group of aspartic acid or glutamic acid.
As such a metabolic system controlled by phosphorylation-dephosphorylation, known well are control system and decomposition system of glycogenesis. This metabolic system is mainly cascade-controlled and regulated by phosphorylation-dephosphorylation.
In recent years, it has been clear that this phosphorylation-dephosphorylation plays important roles in metabolic systems related to diseases.
For example, abnormalities in phosphorylation-dephosphorylation is assumed to contribute to canceration of cells. That is, progress and interruption of cell cycles are controlled by phosphorylation or dephosphorylation of various enzymes (proteins), cyclin and cyclin-dependent kinase (CDK) participates in this phosphorylation or dephosphorylation. In case where this mechanism is damaged, phosphorylation or dephosphorylation is damaged. As the result, aberrant cell growth is aroused.
It has also been clear that protein kinase C participates in degranulation of histamine leading to allergic diseases such as atopic dermatitis and pollinosis, and that phosphorylated tau protein participates in neurofibrillary tangle generated in brain of Alzheimer disease patient.
In view of the above, understanding of situation of phosphorylation-dephosphorylation of proteins should be useful not only for investigation of genetic expression of living body tissue cell, or evaluation of enzyme activity, but also for diagnosis and medical treatment of diseases.
However, conventionally used methods of determination of phosphorylated or dephosphorylated proteins have various defects.
For example, enzyme immunoassay has advantage that it can analyze a very small amount of subject protein sample, but it is difficult to obtain a sufficient amount of necessary antibodies. In addition, when the molecular weight of a subject protein is not more than several kDa, an antibody binding to a phosphorylated site in protein cannot be prepared.
Additionally, detecting a specific binding of phosphoric acid to protein using phosphoric acid labeled by a radioisotope 32P is a possible method. However, handling of radioisotopes deservingly needs special care, and the use of radioisotope requires management and disposal of waste fluid.
Furthermore, since phosphorylated proteins and dephosphorylated proteins have a different electric charge respectively, application of two-dimensional electrophoresis may also be a possible. However, especially in analysis of biological materials, various kinds of proteins included in a sample will make identification of spots very difficult. If radioisotope is used for this spot identification, the above-mentioned problems arise.
In addition to the above-described technologies for determination of phosphorylated or dephosphorylated proteins, a technique using Surface Plasmon Resonance (hereinafter referred to as “SPR”) has been developed as general techniques for investigation for compounds such as protein specifically binding to particular compounds such as ligands (refer to FIG. 1). This technique will, hereinafter, be described in detail.
When a light is totally reflected at an interface between materials having different refractive indexes, a light called evanescent wave is generated on a total reflection surface. In addition, a kind of compressional wave of electron generated in a metal-dielectric substance interface called surface plasmon is generated on a surface of a metal. When an angle of incident light is controlled so that phase velocities of both of evanescent wave and surface plasmon may be coincident, this surface resonance will be resonantly excited, and thereby this surface plasmon can significantly increase an electromagnetic field of the surface of the metal. In this case, since energy of the incident light is absorbed by excitation of the surface plasmon, a strength of a reflected light decreases.
A degree of the angle of incidence and a wavelength of incident light giving this absorption exhibit significantly sharp variation according to a state of the surface of the metal, especially within several hundreds nanometers. Accordingly, variation of states such as existence of compounds on a surface of a metal sensitively affects strength of reflected light. When a ligand or the like is bound on the surface of the metal and then a subject sample is subject to the metal, existence of compounds having interaction with the ligands or the like varies the strength of the reflected light. Therefore, when a ligand or the like is supported on the surface of the metal as shown in FIG. 1, and strengths between reflected lights with and without addition of a subject sample is evaluated, a judgment of existence of compounds having interaction with respect to the ligand or the like can be attained. It is also possible to apply the technique to bio-imaging. That is, in cells or living body tissues, this technique will enable an image of localization of a compound having interaction with respect to a specific compound.
In Japanese translation of PCT international publication No. Hei 11-512186, an example of the technique is described. According to the document, when a running buffer→a subject sample→a running buffer are sequentially subject to a noble metal film as shown in FIG. 1, a degree of the angle of incidence giving SPR shows a variation with time as shown in FIG. 2. The technique of the document measures this variation with time, and examines a minimum and maximum value of reflectance, and a relationship between refractive index and period of time. The document also indicates that there can be determined a dissociation constant and a association constant between a compound supported on the noble metal film and a compound in the subject sample, and a concentration of the compound in the sample.
Further, Japanese translation of PCT international publication No. Hei 10-505910 discloses a technique for performing SPR measurement. In the technique, N-(5-amino-1-carboxy-pentyl)-iminodiacetate is bound to a noble metal film via carboxy methylated dextran, nickel is further coordinated thereto, and SPR is measured. Since this nickel complex shows specific affinity with respect to a peptide having two adjacent histidine residues, it is referred to as His-tag, allowing detection of a peptide having dihistidine residue in a subject sample.
In addition, for measuring a surface plasmon resonance, application of Raman spectroscopy is a possible. Raman spectroscopy is a technique of obtaining information on compounds, wherein, in a scattered light generated by irradiation of a monochromatic light with a fixed frequency ν0 to a material, a scattered light (Raman scattering light) (ν0±νi) other than a same frequency is measured. In detail, since this Raman frequency νi is equal to a frequency between levels of vibration or energy of rotation of a molecule or a crystal constituting the material, this provides sources of information for determination, identification, and quantification of energy levels of the material (refer to “Dictionary for Chemistry (Kagaku Daijiten)” Tokyo Kagaku Dojin).
However, since this Raman scattering light is very weak, its measurement has been conventionally carried out by enhancing the light using the surface plasmon resonance effect (for example, publication of unexamined patent application No. Hei 9-257578). Although light does not couple with electron wave (plasmon) in general, it can couple on a metallic particle surface. Then, the technique enhances the weak Raman band strength with a metal existing in close proximity to a specimen to be measured.
However, the enhancing effect of Raman band strength by the conventional technology was not necessarily satisfactory. The reason is that all target molecules are not forced to contact metal even when a substrate (metal) 2 and a sample 3 were made to exist close to each other as in FIG. 1 of No. Hei 9-257578, although a smaller distance between a target molecule and a metal gives a higher surface plasmon resonance effect. A same circumstance is observed when the metal is added in an aqueous solution sample.
By the way, Hironori Takeda et al., RAPID COMMUNICATIONS IN MASSSPECTROMETRY, Vol. 17, Issue 18, pp. 2075-2081 (2003), describes a method for determining a molecular weight of a phosphorylated compound using a compound specifically bound to a phosphate group, that is phosphoric acid monoester group. However, the document provides neither description nor suggestion for a technical idea of making a metal approach a target compound via this specific binding compound, or for an idea of applying the compound to measurement of a surface plasmon resonance.